A diverse range of inflammatory dermatoses are characterized by vesicles or bullae. Vesiculobullous diseases can be associated with significant morbidity and mortality rates that necessitate their early clinical recognition and prompt management. While the diagnosis and management of vesiculobullous dermatoses may appear daunting to clinicians, we present an updated review regarding the scope of vesiculobullous disorders in detail, including more recent developments in diagnostic testing and targeted therapy [1].

Vesiculobullous lesions present protean manifestations. It may present as formation of vesicles or bullae. However, it is uncommon to appraise vesicles or bullae intraoral as they rupture very easily due to masticatory forces and appear as ulcerations and erosions [2]. Vesiculobullous lesions include viral diseases, autoimmune mucocutaneous diseases due to immunologically mediated mechanism and genetic susceptibility. Clinical history and general physical examination play a vital role which includes the presence of vesicles and bullous in any other region of the body like skin, genitilia and eyes as they may present with dermatological manifestations. Due to indistinguishable appearance of the lesions, the diagnosis can be made histopathologically, clinically and immunological methods. Clinical examination involves a thorough examination of the exposed and mucosal surfaces of the body. The oral diagnostician therefore should have a thorough knowledge of the different procedures that can be used for scrutinising the lesions. This chapter aims to explain various diagnostic tests that can be done for the diagnosis of vesiculobullous lesions [3].

1. Nikolsky Sign

It was first described by Piotr Vasiliyevich Nikolsky (1858 1940) a Russian dermatologist. He related how, after rubbing the skin of patients who had pemphigus foliaceous, there was a blistering or denudation of the epidermis with a glistening, moist surface underneath. According to his explanation, the skin showed a weak relationship and contact between the corneal and granular cell layers on all surfaces and even in places between lesions (e.g. blisters, excoriations) on seemingly unaffected skin. Nikolsky’s observations were later confirmed by Lyell in 1956, who described a Nikolsky's sign in patients with toxic epidermal necrolysis [4].

It is characteristically seen in intraepidermal bullous disorders; whereas in subepidermal VB diseases, the sign is generally absent. Nikolsky’s sign is classically associated with pemphigus vulgaris. However, other blistering conditions are also known to exhibit this sign including pemphigus foliaceous, paraneoplastic pemphigus, oral lichen planus, mucous membrane pemphigoid, bullous pemphigoid, epidermolysis bullosa, Stevens–Johnson Syndrome, Staphylococcal scalded skin syndrome (SSSS), toxic epidermal necrolysis (TEN), linear IgA disease, lupus erythematous (LE), dermatomyositis, chronic erythema multiforme and graft-versus-host disease. This test is difficult to produce in the oral cavity as the blisters and vesicles rupture very early. A Nikolsky sign in the oral cavity is said to be positive when tissue ulceration or blistering is seen after applying mucosal pressure either by blowing air or using a blunt instrument or finger. Principle: In patients with active blistering, firm sliding pressure with a finger separates normal appearing epidermis, producing erosion. Method: This is done by applying lateral pressure with the index finger which provides the shearing force to disrupt the intercellular adhesion in clinical Nikolsky’s sign. If the weakening of the intercellular adhesion is present but not marked, then the same shearing force may produce minimal damage at the cellular level which can be demonstrated only microscopically. As microscopic Nikolsky’s sign sometimes spans only a few cells, serial sections may be required to avoid missing a cleavage [5].

2. Tzanck Test

George Papanicolaou is considered the father of exfoliative cytology, but cytology was first used in cutaneous disorders by Tzanck in 1947, for the diagnosis of VB disorders, particularly herpes simplex. Since then cytology has been widely used by dermatologists for diagnose.

Tzanck smear is a very simple and rapid technique. For viral infections, samples should be taken from a fresh vesicle, rather than a crusted one, to ensure the yield of a number of virus infected cells in various cutaneous dermatosis.

A typical Tzanck cell is a large round keratinocyte with a hyperchromatic nucleus with peripheral condensation of chromatin, hazy or prominent nucleoli, and abundant basophilic cytoplasm. The basophilic staining is deeper peripherally on the cell membrane (“mourning edged” cells) due to the tendency of the cytoplasm to get condensed at the periphery, leading to a perinuclear halo [6].

3. Le Cell Inclusion Phenomenon Or Le Test

This test was first explained by Hargraves for systemic LE (SLE). In tissues, nuclei of damaged cells react with antinuclear antibodies (ANAs), lose their chromatin pattern and become homogenous to produce LE or hematoxylin bodies (amorphous round body in the cytoplasm of the cell). If the serum from a patient suffering from SLE is added to the buffy coat of normal blood, a typical LE cell will develop. LE cell is any phagocytic leukocyte (neutrophils or macrophage) that has engulfed the denatured nucleus of an injured cell and contains an amorphous round body (LE body), serum nuclear globulin (IgG), and complement [7,8].

4. Pathergy Test

Pathergy phenomenon is defined as a state of altered tissue reactivity that occurs in response to minor trauma. Pathergy test (PT) is an easy to perform skin test to look for the pathergy phenomenon. The cutaneous injury leads to inflammatory response that is more prominent and intense than normal saline and shows release of cytokines from keratinocytes in the epidermis or dermis resulting in a perivascular infiltration observed on skin biopsy [9]. A hairless area on the flexor aspect of the forearms is usually chosen as the test sites. After asepsis of skin with 100% alcohol, 20-G needle tips are blunted using the cap and pricked intradermally at a 45°–90° angle with 0.1ML normal saline. Prick sites were evaluated at 48th hour. Papules of >2 mm in diameter and pustules with or without erythematous halo at any prick site is a positive result. Development of only crusts or needle mark due to the trauma or minimal erythema without papule formation is considered negative. Pathergy test is positive in Behcet’s disease [2].

5. Brocq’s Phenomenon

Brocq′s phenomenon is the presence of subepidermal hemorrhage on scraping of the lesion. This is in contrast to the scratching of the surface of lesions of psoriasis, which results in pin-point bleeding [2].

6. Immunofluorescence (If)

IF is an antigen–antibody reaction where the antibodies are tagged (labeled) with a fluorescent dye and the antigen-antibody complex is visualized using ultraviolet (fluorescent) microscope. IF is a well-established technique used for detection of wide variety of antigens in tissues or on cells in suspension. Coons developed IF in 1940 with blue fluorescing compound, beta anthracene. The current gold standard of diagnostic testing for autoimmune blistering skin diseases is DIF microscopy to demonstrate tissue-bound autoantibodies and/or of C3 in the patient’s skin or mucous membrane.

7. Immunofluorescent Techniques

Direct Immunofluorescence DIF is a one-step procedure that involves application of fluoresceinated antibodies to a frozen section of the skin. DIF is diagnostic in pemphigus, pemphigoid, gestational pemphigoid, dermatitis herpatiformis, linear IgA bullous dermatosis, and epidermolysis bullosa acquisita [3].

8. Direct Immunofluorescence

It is the gold standard test which will clinch the diagnosis of AIBDs by identifying the antibodies that are bound in vivo to the tissue antigens in biopsy specimen. Details of the technique are depicted in. Interpretation of direct IF (DIF) results is based on five features: the primary sites of immune deposition (e.g., ICS or BMZ), class of immunoglobulin, pattern of immune deposit (linear or granular), number of immune deposits, and deposition in other sites besides the main site [4]. With the use of these parameters, a pattern approach can lead to an accurate diagnosis in majority of specimens (Fig 1).

9. Indirect Immunofluorescence

IIF is a two-step serological technique for the detection of circulating antibodies. Patient's serum is diluted in serial dilutions (starting at 1:10) with phosphate-buffered saline and incubated with a suitable substrate. The sensitivity of IIF is generally low when compared to DIF and depends on the substrate used. However, the sensitivity can be increased using combination of substrates, for example, normal human skin (NHS) and monkey esophagus (MO) in pemphigus.10 It has been shown that a calcium-containing buffer solution enhances the sensitivity of IIF in pemphigus group of patients by two or more doubling dilutions (Fig 1).

Figure 1: Diagram Showing Important Steps in the Direct Immunofluorescence Test & Indirect Immunofluorescence.

1. Complement Fixation

This technique is obsolete now, especially with the availability of ELISA. This complex technique was used to identify circulating complement-fixing antibodies in pemphigoid gestationis (PG). Circulating complement-fixing antibodies are an immunopathological hallmark of PG and are seen along the DEJ by IIF microscopy on human skin after preincubation with a complement source [4].

2. Modifications in Direct Immunofluorescence

Outer root sheath of anagen hair is structurally analogous to the epidermis and hence may contain pemphigus antigens. DIF of anagen hair has shown ICS staining in 85% of patients with pemphigus. DIF of Tzanck smear has also been studied and found to be useful in pemphigus [4].

3. Salt-Split Technique

This technique involves incubation of skin in 1M sodium chloride for 48-72 h and subsequently splitting the skin between the epidermis and dermis at the level of lamina lucida. In our laboratory, we prefer to incubate the skin substrate in a vial containing 1 M sodium chloride in a rotator at 4 0C in a refrigerator. After overnight incubation, the skin substrate is gently teased to separate epidermis from dermis. This simple technique is extremely useful to subcategorize subepidermal immunobullous diseases (SIBDs); few antigens will remain on the epidermal side (‘roof’) of the split while others will relocate to the dermal side (‘floor’).  Based on the location of BMZ band with respect to the split, SIBDs may be classified into “roof-” or “floor-” binding pattern. For example, BP and inflammatory form of epidermolysis bullosa acquisita (EBA) shows clinicopathological overlap and DIF is indistinguishable in these two conditions. However, salt-split technique (SST) helps distinguish these two conditions as antibodies in BP are “epidermal” binding while EBA shows “dermal-” binding antibodies. It can be performed on the patient's skin (direct SST) or using NHS which is then incubated with patients’ serum (indirect SST). Indirect SST is preferred as it is easier and is similar in accuracy to direct SST [4]. Direct SST is done in cases where IIF tests are negative (Fig 2).

Figure 2: Salt-Split Study.

1. Use of Biochip Mosaic Slides

BIOCHIP mosaic slides have been found to be useful in screening autoantibodies in AIBDs patients. These ready to use slides are available commercially and contain six different substrates (monkey esophagus, primate salt-split skin, recombinant BP180 NC16A, membrane-bound Dsg 1 ectodomain, Dsg 3 ectodomain, and the C-terminal globular domain of BP230) in a miniature field [10]. Technically, this is a modified IIF wherein serum from patients with suspected AIBD is added to these slides and examined under fluorescence microscopy. The advantage of this technique is that it is a useful tool to screen autoantibodies in AIBDs as well as to identify the target antigen. This technique avoids the need to take frozen sections of a suitable substrate. Thus, BIOCHIP mosaic is a simple, standardized, and readily available novel tool that will further facilitate the diagnosis of AIBDs. Validation of the BIOCHIP showed high specificity and high sensitivity for PV, pemphigus foliaceus (PF), and BP [4].

2. Enzyme-Linked Immunosorbent Assay

In recent years, recombinant and cell-derived forms of the target antigens have been applied in the development of sensitive and specific ELISA for the detection of circulating autoantibodies. The commercially available ELISA plates are precoated with recombinant antigen. So far, ELISA systems are available for the diagnosis of pemphigus and pemphigoid diseases [11].

ELISA system has several advantages. It is a quantitative method for measuring specific circulating antibody levels, similar to IIF. Hence, ELISA is useful both in the diagnosis as well as to monitor the disease activity in AIBD such as pemphigus. However, unlike IIF which is observer dependent, ELISA plates are read automatically and results do not depend on the observer. The procedure is quite simple; several samples can be tested simultaneously and the results can be obtained rapidly. It is useful to distinguish PF (anti-Dsg 1 antibodies only) from PV (anti-Dsg 3 with or without anti-Dsg 1 antibodies, depending on the clinical phenotype of PV). Disadvantage of ELISA is that it may not detect all cell surface antibodies involved in the pathogenesis of AIBD, whereas the IIF method may detect antibodies directed against a variety of other nonpathogenic antigens present in normal epithelium. For example, negative result for Dsg 1 and 3 does not rule out the possibility of other rarer subtypes of pemphigus with antibodies to other antigens as in case of PNP and IgA pemphigus. Hence, ELISA should be used as a complementary test to IIF and not as a substitute [4].

3. Immunoblotting and Immunoprecipitation

These techniques provide information on the molecular weight of the target antigen in AIBDs. The antigen source for IB is epidermal or dermal extracts, whereas cultured keratinocytes are the antigenic source for immunoprecipitation (IP). The proteins (antigens) are separated according to their molecular weight by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and the gel is then transferred to nitrocellulose paper. Serum to be tested is then added to nitrocellulose paper and development of a band at a particular molecular weight indicates the target antigen which can be easily inferred by matching it with a control. For example, if the serum from a patient with a SIBD labels a dermal protein of 290 kDa, the target antigen is likely to be Type VII collagen and the diagnosis is EBA. However, these techniques are time-consuming, labor-intensive that require skilled personnel and can realistically be performed only in specialized centers. Most IP techniques involve the use of radioisotopes, limiting their practical use. In addition, SDS destroys the conformational epitopes, and hence, IB is not useful in the diagnosis of pemphigus since most epitopes on Dsg 1 and 3 are conformational [4].

4. Immunoelectron Microscopy

Immunoelectron microscopy (IEM) demonstrates the precise ultrastructural location of antigens in AIBDs. It is akin to DIF and IIF except that visualization of antibodies involves the use of electron-dense gold-labeled antibody probes. When the gold labeling colocalizes with a known protein, the antigen can be indirectly inferred. IEM can be carried out in the same biopsy sample that has been sent for DIF in Michel's medium [4].

Figure 3: Interpretations of Various Disease.

There still remains a dilemma in diagnosing autoimmune VB disease. With the advancement of molecular technology, newer techniques like immunoprecipitation, Western blot analysis, and ELISA have evolved and gradually being used in the domain of immunobullous diseases. However, these investigations are complex, expensive, and more time consuming. IF still remains the gold standard in diagnosing VB lesions as it is simple, reproducible, and less time consuming technique.

Conflict of Interest

The authors declared that there is no conflict of interest.

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